Applications of CRISPR-Cas Technology in S. thermophilus Bacterial Adaptive Immunity

Applications of CRISPR-Cas Technology in
S. thermophilus Bacterial Adaptive
Immunity

Application of CRISPR-Cas Technology
Lay out Summary
CRISPR stands for clustered regularly interspaced short palindromic repeats while Cas stands for
CRISPR-associated protein. CRISPR is a group of DNA chain that can be established within the
genes of prokaryotic creatures such as archaea and bacteria. The sequences involved are imitated
form DNA portions form viruses due to which prokaryotes are infected before and are used to
expose and shatter DNA from alike viruses all along consequent infections.
Analyzation of genes with the help of CRISPR CAS
CRISPR CAS is used to analyze genes with creatures.
Cas(termed as CRISPR-associated protein) is a type of enzyme CRISPR progressions as a
mentor cleave and recognize definite strings of DNA equivalent to CRISPR progressions. Cas
combines with CRISPR sequences in order to form the origin of a mechanism that is called
CRISPR-Cas9.
Together CRISPR-Cas constitutes a system termed as a prokaryotic immune system which
deliberates opposition to external genetic elements including those existing within phages and
plasmids which supports a system of acquired immunity.
Application in various fields
CRISPR is a very huge and widely used technology that has its application in various fields.
More than 1000 research paper had been published mentioning CRISPR. It is used to activate
static or unused genes of the human body, used to analyze ‘candida albicans’, to transform yeast,
etc.
Theme
The theme of the above research is that it focuses on putting the light on those areas where there
is a huge application of CRISPR CAS system, whether it is used in transforming yeasts or
altering genes.
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Aims and objectives
● To analyze various types of gene editing
● To study about an architecture of S. thermophilus CRISPR 3 and S. pyogenes type II CRISPR-
Cas loci
● To examine a novel Streptococcus thermophilus locus which is termed as CRISPR 3
● To provide a new method in relation to CRISPR CAS technology
Timescale
Main Activities/ Stages
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6
Topic selection and its scope 
Secondary data sources
identification

Research proposal preparation 
Preparation of literature review 
Description of research methodology 
Analysing data 
Findings comparison 
Conclusion and recommendations  
Finalising and submission  
S. Thermophilus under CRISPR CAS
CRISPR-Cas locus provides a particular study about the immunity against external components
which are more variable amidst various prokaryotes. The main purpose of the above research is
to focus on terminating the editing of genome protocols found in Streptococcus thermophilus. At
first, those protocols and conditions are introduced that play a role in genome editing. Moreover,
categorization and variety related investigation S. thermophilus CRISPR-Cas help in
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understanding the connection among Streptococcus. Also, its ability to differentiate external
segments and spacer source investigation also signifies a new way of phase opposition.
The system of CRISPR-Cas was brought into being in thermophilic archaea for the very first
time and was utilized to combat external infections related to DNA (Garneau et al., 2010). It
constitutes Cas and CRISPR protein, the anterior is a part of a common group of short-layoff
palindromes which consist of various duplicative progressions and the following one is a type of
protein which is correlated with helicase activity, nuclease, and CRISPRs genes (Chylinski et al.,
2014). The structure of the system of CRISPR-Cas is more alike RNA interference. There are a
total of three stages included in its immune system namely interference, adaption and expression
(Choi and Lee, 2016). Moreover, the new system of classification that mainly includes 3 types
was suggested by Makarova et al. (2011).
Background
CRISPR CAS is a widely used/applied technology which helps in many ways to cure some
serious diseases including cancer, or altering genes. The above research focuses on providing a
context to the use of S. Thermophilus with the help of CRISPR CAS system. CRISPR CAS
changed the history of revolution after its foundation and now it plays a major role in genome
editing.
Themes
Use of S. Thermophilus
A very important and most beneficial lactic acid bacteria termed as Streptococcus thermophilus
is broadly adopted in the generation of dairy products. Additionally, its use in the field of
CRISPR Cas system appears to be one more popular area of research. After the continuing
process of evolution and immunity, S. thermophilus CRISPR Cas loci provides abundant
varieties. In accordance with dissimilar genetic location, duplicative progressions, and Cas
genes, they are divided into 4 major types- CRISPR 1, CRISPR 2, CRISPR 3, and CRISPR 4.
All of these are further enclosed in 3 types of CRISPR Cas system. The sequences of the
CRISPR Cas system differ from part to part due to the reason that there is a difference in the
amount of space.
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Application of S. thermophilus CRISPR Cas system in the field of gene editing
When the CRISPR system was compared with intensely small recombineering adeptness which
is assumed to be 10-4 to 10-6 (Sharan et al., 2009) and tedious expansion of classical methods of
homologous recombination, it could undoubtedly clarify the mutant architecture due to the
reason that it has high efficiency of editing (nearly about 50 to 100%) and chances to elite dual-
crossover cases in single step (Chen et al., 2017).
The start of the CRISPR Cas system is from progression definite identification and object DNA
loci cleavage. The termination of the system is with permanent mutations over DNA restoration
system (Selle and Barrangou, 2015). Currently, the system of CRISPR-Cas associated with S.
pyogenes (SpCas9) is broadly enforced as an appliance in gene alteration. Nevertheless, weak
points of the above system are recorded as its additional studies and considerable application
(Hsu et al., 2013). Due to the same reason, the focus of researchers is more on the S.
thermophilus CRISPR Cas system that provides inferior targeting capability and lesser Cas
portion (Xu et al., 2015). In the opion of Müller et al., (2016) the main process of this system
contains the building of working vector that contains the progression of StCas9 system and
interpretation analysis in the host cell.
Basis of similarities between StCas9 and SpCas9
Various researches propose that the cleavage activities shown by StCas9 system are very similar
to that of SpCas9 system and workability of gene editing in plants, human and microorganisms
(Xu et al., 2015; Müller et al., 2016; Steinert et al., 2016). The Cas9 protein is one of the
essential parts of the StCas system. As the director of probing action, the compositions of Cas9
include a recognition(REC) lobe and a nuclease (NUC) (Anders et al., 2014; Nishimasu et al.,
2014). Before, the PAM-definite probing of destination DNA dual strand is mainly due to RuvC,
NUC alike nuclease and PI domains. And the further is accountable for identification of sgRNA
destination DNA complex (Chen et al., 2017). St1Cas9 and St3Cas9 are included in the StCas9
system that further present image of S. thermophilus CRISPR Cas system in CRISPR 1 and
CRISPR 3 loci respectively. St1Cas9 addresses 5′-NNAGAAW while St3Cas9 addresses 5′-
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NGGNG (Garneau et al., 2010; Magadán et al., 2012). Thus, the destination capability shown by
both the system is lightly dissimilar. The efficiency presented by St3Cas system is higher than
the efficiency presented by St1Cas system (Müller et al., 2016).
Nevertheless, both the systems can be utilized in the modification of genome in accordance with
existing researches (Xu et al., 2015; Müller et al., 2016; Steinert et al., 2016).
Factors affecting gene editing
In addition, there are various important factors that affect editing capability and activities of
StCas9 system. First of all, there is sgRNA that consist of tracrRNA (trans-activating RNA) and
crRNA (CRISPR RNA) that possesses its usual stem-loop architecture which can improve the
synergy among sgRNA and Cas9. But ring and stem have authority over its action. For example,
a stem-circle structure in 3′ end of tracrRNA assumes crucial jobs in action of StCas9. What's
more, its inward lumps are fundamental for the elements of StCas9 while the difference inside
circle arrangements has no impact. (Xu et al., 2015). As noted, crRNA is the structured spacer
following up on the target DNA strand to give explicit focusing on. tracrRNA is in charge of
Cas9 blend and its 3′ locale is fundamental to StCas9 action (Wiedenheft et al., 2012; Xu et al.,
2015). Moreover, the measurements of sgRNA and Cas9 is firmly identified with the askew
effectiveness of StCas9 framework while abbreviated tracrRNA could raise its indicating
capability. Also, PAM and its close-by bases are critical for explicit acknowledgement of target
pieces. Particularly, G in PAM a respectable halfway point is fundamental for the action of
StCas9. Taking everything into account, StCas9 framework cannot exclusively be generally
utilized in the editing of genes yet in addition present increasingly dynamic application
prospects.
New method for amplification of products
Moreover, a new method termed as CRISPR array assembling method which is based on spacer
S. thermophilus has been described. Along these lines, this can be utilized as a leap forward of
helpful multiplex focusing on. First of all, tracrRNA tape, cas9 part, and CRISPR pioneer
arrangement together with CRISPR eliminator ought to be gotten by amplifying of PCR.
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